1. Field of the Invention
The present invention relates to a composition for improving the stability of bacteriophage originated lysin proteins greatly even when the composition contains a high concentration of the bacteriophage originated lysin proteins. More precisely, the present invention relates to a method and a composition for improving significantly the stability of SAL-1 or LysK, the bacteriophage originated lysin protein, included at a high concentration in the composition.
2. Description of the Related Art
Since 1990s, the resistant bacteria showing the resistance against many antibiotics which had been widely used for the treatment of infectious diseases have been increased with causing problems. The most serious problem of them is the significantly lowered treatment effect of antibiotics in treating infectious diseases.
Therefore, it is an urgent request to develop a novel antibiotic substance that can overcome the said problem of resistance of the conventional antibiotics. The promising candidate for the novel antibiotic substance that draws our attention most is the bacteriophage originated lysin protein. This is called the bacteriophage lysin protein or lysin protein or lysin. The bacteriophage lysin protein is a kind of enzyme that is generated from the genetic information of a bacteriophage. The biological activity of the bacteriophage lysin protein, that is the enzymatic activity, is to destroy the peptidoglycan layer that is the major structure of bacterial cell wall. The bacteriophage lysin protein is mainly working in the course of destruction of bacterial cell wall. More precisely, when a bacteriophage is infected into a host, it is proliferated therein and the second generation bacteriophages are generated in the host bacteria. Then, the generated bacteriophages attempt to come out of the host bacteria through the cell wall, during which the bacteriophage lysin protein is working to destroy the cell wall (J. Bacteriol. 186: 4808-4812, 2004).
The bacteriophage lysin protein is naturally generated in the inside of bacteria from the genetic information of a bacteriophage, as explained hereinbefore, but is also synthesized by using recombinant protein technology and then applied to the bacterial cell wall in order to break the peptidoglycan layer. Because of this characteristics, the attempts to use the bacteriophage lysin protein as an antibacterial protein working against bacteria have been increased (U.S. Pat. No. 8,058,225; U.S. Pat. No. 8,105,585). In particular, the attempt to use this protein as a treatment agent for infectious diseases caused by the resistant bacteria is focused on a different mode of action from that of the conventional antibiotics (Science 294: 2170-2172, 2001; Curr. Opin. Microbiol. 8: 480-487, 2005).
SAL-1 that has been developed by the present inventors is also one of the bacteriophage lysin proteins (Antimicrob. Agents Chemother. 55: 1764-1767, 2011). SAL-1 comprises the amino acid sequence represented by SEQ. ID. NO: 1 and has the bacteriolytic activity specific to Staphylococcus aureus. In particular, SAL-1 also displays the bacteriolytic activity against the antibiotic-resistant MRSA (methicillin-resistant Staphylococcus aureus) or VRSA (vancomycin-resistant Staphylococcus aureus). Thus, it can be used as a treatment agent for infectious diseases caused by MRSA or VRSA. The said MRSA and VRSA are the representative antibiotic-resistant bacteria and the number of death caused by the infection with these is very big world-widely.
SAL-1 is very similar to LysK having the amino acid sequence represented by SEQ. ID. NO: 2 and the difference is found only in three residues. However, the antibacterial activity of SAL-1 is almost double the activity of LysK (Antimicrob. Agents Chemother. 55: 1764-1767, 2011).
To use the bacteriophage lysin protein commercially, it needs to be prepared in the form of a high concentration formula. Particularly, when it is used as a medicine, a high concentration unit is advantageous for the administration and handling because when a unit contains a high concentration of the protein, the dosage can be reduced.
In the previous study, the present inventors found out that when the said SAL-1 and LysK were included in a solution at a high concentration, aggregation was observed over the time of storage and this aggregation was also accelerated by an external physical impact, suggesting that the stability of the solution was in question. That kind of disadvantage was not preferred for the industrial use of the lysin protein. To secure the stability during the storage and for safe handling, it was required to develop a method to provide the stability high enough to a composition even when it is prepared in a high concentration liquid form.
The present inventors have confirmed that the addition of calcium ions or magnesium ions to the lysin protein is effective in increasing the biological activity thereof. However, even though the addition of such divalent cations was effective in increasing the biological activity, it also caused the decrease of the stability of the lysin protein included in the liquid form composition. To use industrially the composition comprising these two lysin proteins at high concentrations as active ingredients, it is also requested to develop a method to secure the stability of the lysin protein in the presence of calcium or magnesium ions.
Numbers of research papers and patent documents have been cited in this invention, which are presented in the brackets. At this time, the cited papers and patent documents are included in this invention as a whole in order to describe the arts and spirits and scope of the present invention more clearly.